Bordetella pertussis is a bacterial pathogen that colonizes the human respiratory epithelium to cause whooping cough. To obtain nutritional iron, B. pertussis produces the dihydroxamate siderophore alcaligin and also expresses activities required for the utilization of host heme compounds as well as certain non-native siderophores, including the potent and ubiquitous catechol siderophore enterobactin. Genes encoding these three iron-scavenging systems have distinct positive transcriptional regulators that respond to the cognate iron source for maximal expression of the genes required for its utilization. Expression of all three positive regulators is repressed by the global iron starvation regulator Fur, using iron as corepressor. We hypothesize that Bordetella cells establish priorities in iron transport gene expression by sensing and responding to the presence of the available iron source and selectively activating expression of genes involved in its assimilation. The ability of Bordetella species to prioritize the expression of different iron systems may be important for effective adaptation and multiplication in the host environment. The proposed studies will analyze the in vivo importance of each of the three iron uptake systems in animal models of infection and will determine whether each system is functionally distinct or whether they have shared functional relationships. Mechanistic features of siderophore signaling and transcriptional activation will be delineated for the native alcaligin siderophore system and the enterobactin siderophore utilization system. The importance of the ability to transcriptionally respond to the appropriate iron source in vivo will be evaluated using Bordetella mutants producing novel hybrid regulators with reversed inducer and target gene specificities. A cell surface signaling phenomenon uniquely involved in regulation of the Bordetella host heme-iron utilization system will be investigated, and interacting signaling and regulatory protein domains will be defined. Spatiotemporal analysis of in vivo expression of the three iron systems will determine which systems are operational in the animal host and assess whether the systems are differentially expressed in certain tissue sites or during distinct stages of infection. Because B. pertussis is an obligate human pathogen with no known environmental or nonhuman animal reservoirs, it represents an ideal model organism for analysis of the host-parasite relationship and the physical, chemical and innate biological conditions that impact on the growth of bacteria in a host environment.